The present invention is concerned with stable, non-immunogenic, physiologically readily compatible, dissolved or lyophilised galenical preparations of human proteins and especially of erythropoietin, as well as processes for the preparation thereof.
Human proteins are proteins characteristic of the body which only occur in small amounts, for example, tissue plasminogen activator (tPA), streptokinase, urokinase, interferon, various colony stimulating factors (CSF) and erythropoietin (EPO). The present invention will be explained in more detail using EPO as an example which is preferably used in the formulations.
Erythropoietin (EPO) is a glycoprotein which stimulates the formation of haemoglobin or erythrocytes in the bone marrow. This lipoprotein is mainly formed in the kidneys but is found in very small amounts in the serum and, under physiological conditions, is partly excreted in the urine.
The absence of EPO in the case of kidney insufficiency also causes a renal anaemia. By the administration of EPO in physiological amounts, i.e., a few micrograms, in one or more dosings, the formation of erythrocytes can again be stimulated. Since the body reacts sensitively to small dosage changes, the dosing must be exactly reproducible. EPO is usually injected as an aqueous solution either intramuscularly or intravenously or is administered as a spray via the nasal mucous membrane.
However, it is known that EPO, not only the product first obtained from human urine (Mijake et al., J. Biol. Chem. 25, 5558-5564 (1977) but also the product more recently produced by gene technology (WO 85-02610), is not stable in aqueous solution and, even in the case of storage at -80.degree. C., comparatively large activity losses occur. These two known products differ somewhat in their glycosilation pattern and in their activity; a direct comparison with the EPO contained in the serum has hitherto not been known.
These activity losses are to be attributed, on the one hand, to a destruction of the EPO by catalytic effects of the surface of the ampoules used for storage due to traces of heavy metals, atmospheric oxygen and the like, and also, on the other hand, to a deposition of EPO molecules on the vessel wall, a partial denaturing thereof possibly also taking place. Since, as stated above, only a few micrograms are present in each dosage unit, the losses due to adsorption can be considerable, even after a short storage time.
Therefore, European Patent Specification No. 0,178,576 describes the inhibition of this deposition on the vessel wall by the addition of polymeric compounds, such as human or bovine serum albumin, lecithin, dextran, cellulose, polyethylene glycol, and the like, thereby making possible an EPO level of 75 to 98% after about 2 hours storage at 20.degree. C., compared with only 16% without such an addition. However, there was only measured the level of a radioactive labelled (.sup.14 C) product so that these experiments say nothing about the stabilisation of the EPO against decomposition.
However, according to our findings, a long-term stabilisation with such agents cannot be achieved, i.e., the EPO effectiveness in the mouse test decreases strongly and, in addition, these agents can bring about immunogenic reactions when injected.
Furthermore, in European Patent Specification No. 0,178,665 there are disclosed "stabilizers", especially for lyophilised EPO preparations. Besides the polymeric substances PEG 4000, gelatine and dextran 40, there are mentioned various sugars and sugar alcohols, amino acids, inorganic salts and thiol compounds. Combinations of these materials with human serum albumin, gelatine and dextran are also mentioned. In this literature reference, too, there is determined only the level of radioactivities after 2 months storage of the lyophilised products. This is given as being 87 to 99%, in comparison with 60% without any addition. Since the lyophilised material was used directly after the production as a standard, it is not stated how high the activity losses are in the case of the production of the preparations. These preparations also display a high effectiveness loss in the mouse test.
Therefore, there exists the problem of finding a readily compatible EPO preparation which is storage-stable, i.e., ensures the in vivo effectiveness, does not lead to adsorptions on the ampoule and syringe walls, and can easily be made into an injectable form.